Evaluation of the antimicrobial and cytotoxic potential of endophytic fungi extracts from mangrove plants Rhizophora stylosa and R. mucronata

Mangrove endophytic fungi are tolerant to numerous stresses and are inevitably capable of exhibiting excellent biological activity by producing impressive numbers of metabolites with special biological functions, based on previous work on the biological potential of mangrove-derived endophytic fungi. To obtain marked antimicrobial and cytotoxic fermentation products of culturable endophytic fungi from mangrove forests, our research evaluated the antimicrobial and cytotoxic activities of crude extracts of endophytic fungi from Rhizophora stylosa and Rhizophora mucronata. Forty-six fungal isolates were cultured on four different media, namely, dextrose agar (PDA), Czapek’s agar (CZA), rice medium (RM) and grain medium (GM) and harvested by ethyl acetate solvent at 40 days. The extracts were tested for antimicrobial activity by the microdilution method against the gram-negative bacteria Pseudomonas adaceae (PA), gram-positive bacteria Enterococcus faecalis (EF), methicillin-resistant Staphylococcus aureus (MRSA) and pathogenic fungus Monilia albicans (MA). The cytotoxic activity of the extracts was evaluated by MTT assay using A549 human lung cancer cells, HeLa human cervical carcinoma cells, and HepG2 human hepatocellular cells. The results showed that rice medium could promote the secretion of antimicrobial and antitumour secondary metabolites of endophytic fungi in comparison with other cultivation media. Seventeen strains (68%) from R. stylosa exhibited inhibitory effects on indicators, especially N. protearum HHL46, which could inhibit the growth of four microbes with MIC values reaching 0.0625 mg/mL. Fifteen strains (71.4%) from R. mucronata displayed activities against human pathogenic microbes; in particular, Pestalotiopsis sp. HQD6 and N. protearum HQD5 could resist the growth of four microbes with MIC values ranging from 0.015 to 1 mg/mL. In the cytotoxicity assay, the extracts of 10 strains (40%), 9 strains (40%) and 13 strains (52%) of R. stylosa and 13 strains (61.9%), 10 strains (47.6%) and 10 strains (47.6%) of R. mucronata displayed cytotoxicity against A549, HeLa and HepG2 cancer cells with cell viability values ≤ 50%. Neopestalotiopsis protearum HHL46, Phomopsis longicolla HHL50, Botryosphaeria fusispora HQD83, Fusarium verticillioides HQD48 and Pestalotiopsis sp. HQD6 displayed significant antitumour activity with IC50 values below 20 μg/mL. These results highlighted the antimicrobial and antitumour potential of endophytic fungi from R. stylosa and R. mucronata and the possibility of exploiting their antimicrobial and cytotoxic agents.


Results
Antimicrobial activity of fungal extracts. From a total of 46 fungal extracts assayed, 32 extracts (69.6%) showed antimicrobial activity against at least one of the indicator pathogenic microbes tested (Tables 1 and 2). The antimicrobial activity of the same isolated fungal strain was significantly different (P < 0.05) when cultured on different media, but all of the antimicrobial activities were weaker than that of the positive control.
Of the endophytic fungi isolated from R. stylosa (25 isolates, Table 1), RM was determined to be more suitable for antibiotic production in fungal isolates than the other three media (P < 0.05). Of these, 13 strains cultured on RM (52%) exhibited antimicrobial activity using 1 mg/mL extracts, and among them, 7 strains had stronger inhibitory effects on MRSA with MIC values less than 0.5 mg/mL. HHL55 showed the broadest antimicrobial spectrum against four indicator test microorganisms, and CZA culture of HHL55 was found to show the most potent antimicrobial activity against PA with an MIC value of 0.031 mg/mL. Only 2 strains fermented on the GM displayed low inhibitory activity with an MIC value of 1 mg/mL. In addition, three culture media derived from HHL94, HHL64 and HHL82 showed inhibitory effects on three indicator microorganisms.
Of the endophytic fungi isolated from R. mucronata (21 isolates, www.nature.com/scientificreports/ extracts cultured on CZA (10 isolates, 47.6%) and RM (10 isolates, 47.6%) in comparison with PDA (7 isolates, 33.3%) and GM (2 isolates, 9.5%) at the selected concentration of 1 mg/mL (P < 0.05). The RM of 9 isolates and CZA of 3 isolates showed strong inhibitory effects on MRSA with MIC values less than 0.5 mg/mL. Only the extract of HQD20 cultivated on GM inhibited the growth of MRSA and MA. Moreover, HQD6 and HQD5 displayed antimicrobial activity against the growth of four indicator microorganisms tested with MIC values ranging from 0.015 to 1 mg/mL.
In an attempt to promote antitumour substance production, four different media were adopted for fungal isolate cultivation to activate biosynthetic silencing of gene expression. We found that extracts of 1 isolate (HQD28) cultured on PDA, 1 isolate (HQD48) cultured on CZA, 3 isolates (HHL82, HQD52 and HQD6) cultured on RM and 4 isolates (HHL46, HQD48, HQD5 and HQD8) cultured on GM exhibited significant antiproliferative activity against at least one of the tested carcinoma cells with an IC 50 < 20 μg/mL. GM culture of R. stylosa endophytic HHL46 and RM culture of HHL61 and HHL82 were most effective against A549, HeLa and HepG2 cells, with IC 50 values of 14.38 ± 1.84 μg/mL, 23.17 ± 4.26 μg/mL and 14.38 ± 1.84 μg/mL, respectively. The significant difference analysis showed that the inhibition of HeLa cells by the products of endophytic fungi of R. stylosa cultured on RM was stronger than those cultured under the other three conditions (P < 0.05). No significant difference (P > 0.05) was observed between the cytotoxicity for A549 of samples from four cultural media having equal degrees at end of 24 h ( Table 3). The extracts from CZA culture of R. mucronata endophytic HQD48 and RM culture of HQD20 and HQD6 exhibited cytotoxicity towards A549, HeLa and HepG2 cells, with IC 50 values of 4.83 ± 1.61 μg/mL, 14.38 ± 1.84 μg/mL and 9.58 ± 0.01 μg/mL, respectively ( Table 4). The genera Pestalotiopsis and Phomopsis were demonstrated to be rich sources of antitumour secondary metabolites. Notably, RM culture

Profiling of bioactive metabolites by HPLC.
According to the screening for antimicrobial activity and cytotoxicity, we found that 6 endophytic fungi showed strong bioactive abilities: HHL46, HHL50, HQD5, HQD6, HQD83 and HQD48. The difference significance analysis showed that RM was to be the best to produce active metabolites and the product diversity of endophytic fungi on RM was analyzed by HPLC in further. Figure 3 shows the chromatograms of the fermented products of these endophytic fungi at 254 nm. The HPLC analysis results provide a wealth of information, and the hydrophilic compounds were found in the first 10 min. Within 10 to 15 min, the moderately polar components and hydrophobic components were washed out. RM cultures of HQD5 and HQD6 showed more diverse secondary metabolites than other cultures.

Discussion
Infectious diseases, including bacterial infections, pose a serious threat to global health and drug resistance 23,24 .
Unfortunately, there has been resistance detected against every antibiotic on the market, and if antibiotic resistance is not mitigated, pathogenic bacteria will once again become one of the leading causes of mortality, with an estimated yearly death toll of ~ 10 million by 2050 25 . The development of new antimicrobial agents is pointed out as an effective solution to address this problem 26,27 . As a promising source of diverse and structurally unprecedented bioactive natural products, mangrove-derived endophytic fungi are unquestionably important and continuously attract considerable attention 28,29 . In our current study, of 46 endophytic fungal strains investigated,  www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 32 extracts (69.6%) exhibited antimicrobial activity, which is consistent with the findings of a previous study by Buatong et al. 30 in which 61.3% of mangrove fungal endophytes produced inhibitory compounds. Cancer-related death is one of most significant threats to human health worldwide, with an estimated 12.7 million new cases and 7.6 million cancer deaths each year 31 . At present, the primary treatment method for cancers is to combine removing the tumour with anaesthetic agents after surgery 32 . Unfortunately, the surgical process possibly leads to tumour progression, causing a large number of tumour cells to be released and reducing the activity of T, B and NK lymphocytes in the postoperative period 33 . It has also generated a large body of information that is being harnessed to develop new therapeutic modalities for treating cancer 34 ; however, the search for cytotoxic agents that selectively impact proliferating cells still plays an essential role in tumour treatment 35 . In our study, of 46 endophytic fungal strains investigated, 23 extracts (50%) showed cytotoxicity, 21 extracts (46.65%) were cytotoxic against A549 cells, 16 extracts (34.78%) were cytotoxic against HeLa cells, and 21 extracts (46.65%) were cytotoxic against HepG cells. This was in accordance with previous reports, in which 9 endophytic fungi were successfully obtained from the leaves of Ginkgo biloba. The extracts of isolates J-1, J-2 and J-3 markedly inhibited the proliferation of HeLa cells, promoted their apoptosis and blocked their migration 36 , and 12 isolate extracts (85.7%) derived from mangrove Rhizophora mucronata were cytotoxic (cell viability < 50%) against T47D cells 12 . In our current study, we found that Pestalotiopsis sp. HQD6 displayed significant antitumour activity with IC 50 values below 20 μg/mL and showed more diverse secondary metabolites than other formulations, which was in accordance with our previous reports that demethylincisterol A 3 was isolated from the R. mucronata endophytic Pestalotiopsis sp. HQD6 and showed significant in vitro cytotoxicity against the human cancer cell lines HeLa, A549 and HepG, with IC 50 values reaching nM ranging from 0.17 to 14.16 nM 19 . It also reported to be a selective inhibitor of the classical nonreceptor protein tyrosine phosphatase Shp2 20 .
Culture-dependent methods have been developed aiming at substantial increases in biologically active secondary metabolite production by any given microorganism 37 . RM was demonstrated to have the highest suitability for antibiotic production; 23 extracts (50%) showed antimicrobial activity against at least one of four strains, and the extract of HQD1 exhibited antimicrobial activity against MRSA with an MIC value of 0.031 mg/ mL, which was in accordance with the previous reports of Rivai et al. 12 . RM (11 isolates, 39.13%) and GM (9 isolates, 19.56%) were more suitable for antitumour agent production, which agreed with previous reports of GM-cultivated Oidiodendron truncatum leading to the discovery of the potent anticancer agent chetracin B with cytotoxicity against five human cancer lines reaching nM degree 38 . The variation in the antibiotic and cytotoxic properties among media could possibly be related to the composition of RM, and GM activated our isolated fungal biosynthetic gene clusters 39 .   The profiling of secondary metabolites by HPLC. To assign the bioactivities of the endophytic fungal cultural extract, we performed profiling of the compounds with a Waters 2998 series HPLC system. Separation was achieved using a 250 mm column at 26 °C with a multistep linear gradient elution program in which chromatographic methanol changed from 0 to 20% in 5 min, from 20 to 25% in 5-15 min, from 25 to 50% in 15-30 min, from 50 to 65% in 30-40 min, from 65 to 85% in 40-5 50 min, and finally from 85 to 100% in 50-60 min. UV spectra were recorded at 254 nm. The extraction of rice medium was used as a control 41 .

Materials and methods
Statistical analyses. SPSS 23.0 software was used for statistical analysis, Ordinary one-way ANOVA and principal component analysis PCA used for the different measured variables in the study. Significance was evaluated in at a level of P < 0.05, for the endophyte extract of different medium to antimicrobial (PCA) and cytotoxic activities (ANOVA) were performed using Tim Duncan's test. All errors are expressed as standard deviations (SD).

Conclusion
To date, there are few systematic studies on the antimicrobial and antitumour potential of mangrove endophytic fungi. Our study indicated that the antimicrobial and cytotoxic activities of mangrove endophytic fungal extracts grown on four media showed distinguishable differences in activities and revealed that RM could promote the secretion of bioactive secondary metabolites. Neopestalotiopsis protearum HQD5 and Pestalotiopsis sp. HQD6 showed potent antimicrobial activity; Neopestalotiopsis protearum HHL46, Phomopsis longicolla HHL50, Botryosphaeria fusispora HQD83, Fusarium verticillioides HQD48 and Pestalotiopsis sp. HQD6 displayed significant antitumour activity. Considering these results, these fungi could be further explored for the characterization of antimicrobial and cytotoxic secondary metabolites, which could explain the significant biological activities of the abovementioned fungal strain.